RESUMEN
Polycomb repressive complex 2 (PRC2) silences gene expression through trimethylation of K27 of histone H3 (H3K27me3) via its catalytic SET domain. A missense mutation in the substrate of PRC2, histone H3K27M, is associated with certain pediatric brain cancers and is linked to a global decrease of H3K27me3 in the affected cells thought to be mediated by inhibition of PRC2 activity. We present here the crystal structure of human PRC2 in complex with the inhibitory H3K27M peptide bound to the active site of the SET domain, with the methionine residue located in the pocket that normally accommodates the target lysine residue. The structure and binding studies suggest a mechanism for the oncogenic inhibition of H3K27M. The structure also reveals how binding of repressive marks, like H3K27me3, to the EED subunit of the complex leads to enhancement of the catalytic efficiency of the SET domain and thus the propagation of this repressive histone modification.
Asunto(s)
Histonas/química , Lisina/química , Complejo Represivo Polycomb 2/química , Dominios Proteicos , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Carcinogénesis/genética , Dominio Catalítico , Cristalografía por Rayos X , Proteína Potenciadora del Homólogo Zeste 2/química , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Lisina/genética , Lisina/metabolismo , Metilación , Modelos Moleculares , Mutación , Oncogenes/genética , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Unión ProteicaRESUMEN
In 2004 an hemagglutinin 3 neuraminidase 8 (H3N8) equine influenza virus was transmitted from horses to dogs in Florida and subsequently spread throughout the United States and to Europe. To understand the molecular basis of changes in the antigenicity of H3 hemagglutinins (HAs) that have occurred during virus evolution in horses, and to investigate the role of HA in the equine to canine cross-species transfer, we used X-ray crystallography to determine the structures of the HAs from two antigenically distinct equine viruses and from a canine virus. Structurally all three are very similar with the majority of amino acid sequence differences between the two equine HAs located on the virus membrane-distal molecular surface. HAs of canine viruses are distinct in containing a Trp-222 â Leu substitution in the receptor binding site that influences specificity for receptor analogs. In the fusion subdomain of canine and recent equine virus HAs a unique difference is observed by comparison with all other HAs examined to date. Analyses of site-specific mutant HAs indicate that a single amino acid substitution, Thr-30 â Ser, influences interactions between N-terminal and C-terminal regions of the subdomain that are important in the structural changes required for membrane fusion activity. Both structural modifications may have facilitated the transmission of H3N8 influenza from horses to dogs.
Asunto(s)
Sustitución de Aminoácidos , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Subtipo H3N8 del Virus de la Influenza A/química , Animales , Cristalografía por Rayos X , Enfermedades de los Perros/genética , Enfermedades de los Perros/metabolismo , Enfermedades de los Perros/virología , Perros , Glicoproteínas Hemaglutininas del Virus de la Influenza/genética , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Enfermedades de los Caballos/genética , Enfermedades de los Caballos/metabolismo , Enfermedades de los Caballos/virología , Caballos , Subtipo H3N8 del Virus de la Influenza A/metabolismo , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/metabolismo , Estructura Terciaria de ProteínaRESUMEN
FAN1 is a structure-selective DNA repair nuclease with 5' flap endonuclease activity, involved in the repair of interstrand DNA crosslinks. It is the only eukaryotic protein with a virus-type replication-repair nuclease ("VRR-Nuc") "module" that commonly occurs as a standalone domain in many bacteria and viruses. Crystal structures of three representatives show that they structurally resemble Holliday junction resolvases (HJRs), are dimeric in solution, and are able to cleave symmetric four-way junctions. In contrast, FAN1 orthologs are monomeric and cleave 5' flap structures in vitro, but not Holliday junctions. Modeling of the VRR-Nuc domain of FAN1 reveals that it has an insertion, which packs against the dimerization interface observed in the structures of the viral/bacterial VRR-Nuc proteins. We propose that these additional structural elements in FAN1 prevent dimerization and bias specificity toward flap structures.
Asunto(s)
Proteínas Bacterianas/química , ADN Cruciforme/metabolismo , Endodesoxirribonucleasas/química , Exodesoxirribonucleasas/química , Resolvasas de Unión Holliday/química , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/metabolismo , Reparación del ADN , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Resolvasas de Unión Holliday/metabolismo , Humanos , Ratones , Datos de Secuencia Molecular , Enzimas Multifuncionales , Unión Proteica , Multimerización de Proteína , Estructura Terciaria de Proteína , Pseudomonas aeruginosa/enzimologíaRESUMEN
Malaria is caused by a protozoan parasite that replicates within an intraerythrocytic parasitophorous vacuole. Release (egress) of malaria merozoites from the host erythrocyte is a highly regulated and calcium-dependent event that is critical for disease progression. Minutes before egress, an essential parasite serine protease called SUB1 is discharged into the parasitophorous vacuole, where it proteolytically processes a subset of parasite proteins that play indispensable roles in egress and invasion. Here we report the first crystallographic structure of Plasmodium falciparum SUB1 at 2.25 Å, in complex with its cognate prodomain. The structure highlights the basis of the calcium dependence of SUB1, as well as its unusual requirement for interactions with substrate residues on both prime and non-prime sides of the scissile bond. Importantly, the structure also reveals the presence of a solvent-exposed redox-sensitive disulphide bridge, unique among the subtilisin family, that likely acts as a regulator of protease activity in the parasite.
Asunto(s)
Calcio/metabolismo , Plasmodium falciparum/enzimología , Proteínas Protozoarias/metabolismo , Subtilisina/metabolismo , Secuencia de Aminoácidos , Animales , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Proteínas Protozoarias/química , Homología de Secuencia de AminoácidoRESUMEN
H10N8 follows H7N9 and H5N1 as the latest in a line of avian influenza viruses that cause serious disease in humans and have become a threat to public health. Since December 2013, three human cases of H10N8 infection have been reported, two of whom are known to have died. To gather evidence relating to the epidemic potential of H10 we have determined the structure of the haemagglutinin of a previously isolated avian H10 virus and we present here results relating especially to its receptor-binding properties, as these are likely to be major determinants of virus transmissibility. Our results show, first, that the H10 virus possesses high avidity for human receptors and second, from the crystal structure of the complex formed by avian H10 haemagglutinin with human receptor, it is clear that the conformation of the bound receptor has characteristics of both the 1918 H1N1 pandemic virus and the human H7 viruses isolated from patients in 2013 (ref. 3). We conclude that avian H10N8 virus has sufficient avidity for human receptors to account for its infection of humans but that its preference for avian receptors should make avian-receptor-rich human airway mucins an effective block to widespread infection. In terms of surveillance, particular attention will be paid to the detection of mutations in the receptor-binding site of the H10 haemagglutinin that decrease its avidity for avian receptor, and could enable it to be more readily transmitted between humans.
Asunto(s)
Aves/virología , Orthomyxoviridae/química , Orthomyxoviridae/metabolismo , Receptores Virales/química , Receptores Virales/metabolismo , Animales , Sitios de Unión , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H1N1 del Virus de la Influenza A/química , Subtipo H7N9 del Virus de la Influenza A/química , Modelos Moleculares , Zoonosis/transmisión , Zoonosis/virologíaRESUMEN
AMP-activated protein kinase (AMPK) plays a major role in regulating cellular energy balance by sensing and responding to increases in AMP/ADP concentration relative to ATP. Binding of AMP causes allosteric activation of the enzyme and binding of either AMP or ADP promotes and maintains the phosphorylation of threonine 172 within the activation loop of the kinase. AMPK has attracted widespread interest as a potential therapeutic target for metabolic diseases including type 2 diabetes and, more recently, cancer. A number of direct AMPK activators have been reported as having beneficial effects in treating metabolic diseases, but there has been no structural basis for activator binding to AMPK. Here we present the crystal structure of human AMPK in complex with a small molecule activator that binds at a site between the kinase domain and the carbohydrate-binding module, stabilising the interaction between these two components. The nature of the activator-binding pocket suggests the involvement of an additional, as yet unidentified, metabolite in the physiological regulation of AMPK. Importantly, the structure offers new opportunities for the design of small molecule activators of AMPK for treatment of metabolic disorders.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Regulación Enzimológica de la Expresión Génica , Adenosina Monofosfato/química , Adenosina Trifosfato/química , Sitio Alostérico , Sitios de Unión , Carbohidratos/química , Dicroismo Circular , Cristalografía por Rayos X , Células HEK293 , Humanos , Interferometría , Fosforilación , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Treonina/químicaRESUMEN
Of the 132 people known to have been infected with H7N9 influenza viruses in China, 37 died, and many were severely ill. Infection seems to have involved contact with infected poultry. We have examined the receptor-binding properties of this H7N9 virus and compared them with those of an avian H7N3 virus. We find that the human H7 virus has significantly higher affinity for α-2,6-linked sialic acid analogues ('human receptor') than avian H7 while retaining the strong binding to α-2,3-linked sialic acid analogues ('avian receptor') characteristic of avian viruses. The human H7 virus does not, therefore, have the preference for human versus avian receptors characteristic of pandemic viruses. X-ray crystallography of the receptor-binding protein, haemagglutinin (HA), in complex with receptor analogues indicates that both human and avian receptors adopt different conformations when bound to human H7 HA than they do when bound to avian H7 HA. Human receptor bound to human H7 HA exits the binding site in a different direction to that seen in complexes formed by HAs from pandemic viruses and from an aerosol-transmissible H5 mutant. The human-receptor-binding properties of human H7 probably arise from the introduction of two bulky hydrophobic residues by the substitutions Gln226Leu and Gly186Val. The former is shared with the 1957 H2 and 1968 H3 pandemic viruses and with the aerosol-transmissible H5 mutant. We conclude that the human H7 virus has acquired some of the receptor-binding characteristics that are typical of pandemic viruses, but its retained preference for avian receptor may restrict its further evolution towards a virus that could transmit efficiently between humans, perhaps by binding to avian-receptor-rich mucins in the human respiratory tract rather than to cellular receptors.
Asunto(s)
Virus de la Influenza A/metabolismo , Gripe Humana/virología , Ácido N-Acetilneuramínico/metabolismo , Receptores Virales/metabolismo , Animales , Sitios de Unión , Aves/metabolismo , Aves/virología , Cristalografía por Rayos X , Glicoproteínas Hemaglutininas del Virus de la Influenza/química , Glicoproteínas Hemaglutininas del Virus de la Influenza/metabolismo , Humanos , Subtipo H7N3 del Virus de la Influenza A/metabolismo , Virus de la Influenza A/química , Virus de la Influenza A/aislamiento & purificación , Modelos Moleculares , Mucinas/química , Mucinas/metabolismo , Ácido N-Acetilneuramínico/análogos & derivados , Ácido N-Acetilneuramínico/química , Unión Proteica , Conformación Proteica , Receptores Virales/químicaRESUMEN
Two classes of antiviral drugs, neuraminidase inhibitors and adamantanes, are approved for prophylaxis and therapy against influenza virus infections. A major concern is that antiviral resistant viruses emerge and spread in the human population. The 2009 pandemic H1N1 virus is already resistant to adamantanes. Recently, a novel neuraminidase inhibitor resistance mutation I223R was identified in the neuraminidase of this subtype. To understand the resistance mechanism of this mutation, the enzymatic properties of the I223R mutant, together with the most frequently observed resistance mutation, H275Y, and the double mutant I223R/H275Y were compared. Relative to wild type, K(M) values for MUNANA increased only 2-fold for the single I223R mutant and up to 8-fold for the double mutant. Oseltamivir inhibition constants (K(I)) increased 48-fold in the single I223R mutant and 7500-fold in the double mutant. In both cases the change was largely accounted for by an increased dissociation rate constant for oseltamivir, but the inhibition constants for zanamivir were less increased. We have used X-ray crystallography to better understand the effect of mutation I223R on drug binding. We find that there is shrinkage of a hydrophobic pocket in the active site as a result of the I223R change. Furthermore, R223 interacts with S247 which changes the rotamer it adopts and, consequently, binding of the pentoxyl substituent of oseltamivir is not as favorable as in the wild type. However, the polar glycerol substituent present in zanamivir, which mimics the natural substrate, is accommodated in the I223R mutant structure in a similar way to wild type, thus explaining the kinetic data. Our structural data also show that, in contrast to a recently reported structure, the active site of 2009 pandemic neuraminidase can adopt an open conformation.
Asunto(s)
Antivirales/farmacología , Farmacorresistencia Viral/genética , Inhibidores Enzimáticos/farmacología , Subtipo H1N1 del Virus de la Influenza A/efectos de los fármacos , Subtipo H1N1 del Virus de la Influenza A/enzimología , Gripe Humana/virología , Neuraminidasa/química , Adamantano/farmacología , Sustitución de Aminoácidos , Sitios de Unión/genética , Cristalografía por Rayos X , Inhibidores Enzimáticos/uso terapéutico , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Subtipo H1N1 del Virus de la Influenza A/genética , Gripe Humana/tratamiento farmacológico , Mutación , Neuraminidasa/antagonistas & inhibidores , Neuraminidasa/genética , Oseltamivir/farmacología , Oseltamivir/uso terapéutico , Pandemias , Conformación Proteica , Zanamivir/farmacología , Zanamivir/uso terapéuticoRESUMEN
An N-terminal fragment of human SHARPIN was recombinantly expressed in Escherichia coli, purified and crystallized. Crystals suitable for X-ray diffraction were obtained by a one-step optimization of seed dilution and protein concentration using a two-dimensional grid screen. The crystals belonged to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 61.55, c = 222.81â Å. Complete data sets were collected from native and selenomethionine-substituted protein crystals at 100â K to 2.6 and 2.0â Å resolution, respectively.
Asunto(s)
Proteínas Portadoras/química , Ubiquitinas/química , Automatización de Laboratorios , Cristalización , Cristalografía por Rayos X , HumanosRESUMEN
SHARPIN (SHANK-associated RH domain interacting protein) is part of a large multi-protein E3 ubiquitin ligase complex called LUBAC (linear ubiquitin chain assembly complex), which catalyzes the formation of linear ubiquitin chains and regulates immune and apoptopic signaling pathways. The C-terminal half of SHARPIN contains ubiquitin-like domain and Npl4-zinc finger domains that mediate the interaction with the LUBAC subunit HOIP and ubiquitin, respectively. In contrast, the N-terminal region does not show any homology with known protein interaction domains but has been suggested to be responsible for self-association of SHARPIN, presumably via a coiled-coil region. We have determined the crystal structure of the N-terminal portion of SHARPIN, which adopts the highly conserved pleckstrin homology superfold that is often used as a scaffold to create protein interaction modules. We show that in SHARPIN, this domain does not appear to be used as a ligand recognition domain because it lacks many of the surface properties that are present in other pleckstrin homology fold-based interaction modules. Instead, it acts as a dimerization module extending the functional applications of this superfold.
Asunto(s)
Proteínas del Tejido Nervioso/química , Pliegue de Proteína , Multimerización de Proteína , Subunidades de Proteína/química , Ubiquitina-Proteína Ligasas/química , Proteínas Sanguíneas/química , Cristalografía por Rayos X , Humanos , Fosfoproteínas/química , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Dedos de ZincRESUMEN
Mdc1 is a large modular phosphoprotein scaffold that maintains signaling and repair complexes at double-stranded DNA break sites. Mdc1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with the tail of γH2AX following DNA damage, but the role of the N-terminal forkhead-associated (FHA) domain remains unclear. We show that a major binding target of the Mdc1 FHA domain is a previously unidentified DNA damage and ATM-dependent phosphorylation site near the N-terminus of Mdc1 itself. Binding to this motif stabilizes a weak self-association of the FHA domain to form a tight dimer. X-ray structures of free and complexed Mdc1 FHA domain reveal a 'head-to-tail' dimerization mechanism that is closely related to that seen in pre-activated forms of the Chk2 DNA damage kinase, and which both positively and negatively influences Mdc1 FHA domain-mediated interactions in human cells prior to and following DNA damage.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Transactivadores/química , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Secuencia de Aminoácidos , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Células Cultivadas , Proteínas Cromosómicas no Histona/análisis , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/análisis , Dimerización , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Fosfotreonina/metabolismo , Dominios y Motivos de Interacción de Proteínas , Treonina/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
SAMHD1, an analogue of the murine interferon (IFN)-γ-induced gene Mg11 (ref. 1), has recently been identified as a human immunodeficiency virus-1 (HIV-1) restriction factor that blocks early-stage virus replication in dendritic and other myeloid cells and is the target of the lentiviral protein Vpx, which can relieve HIV-1 restriction. SAMHD1 is also associated with Aicardi-Goutières syndrome (AGS), an inflammatory encephalopathy characterized by chronic cerebrospinal fluid lymphocytosis and elevated levels of the antiviral cytokine IFN-α. The pathology associated with AGS resembles congenital viral infection, such as transplacentally acquired HIV. Here we show that human SAMHD1 is a potent dGTP-stimulated triphosphohydrolase that converts deoxynucleoside triphosphates to the constituent deoxynucleoside and inorganic triphosphate. The crystal structure of the catalytic core of SAMHD1 reveals that the protein is dimeric and indicates a molecular basis for dGTP stimulation of catalytic activity against dNTPs. We propose that SAMHD1, which is highly expressed in dendritic cells, restricts HIV-1 replication by hydrolysing the majority of cellular dNTPs, thus inhibiting reverse transcription and viral complementary DNA (cDNA) synthesis.
Asunto(s)
VIH-1/fisiología , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/metabolismo , Nucleósido-Trifosfatasa/química , Nucleósido-Trifosfatasa/metabolismo , Regulación Alostérica , Biocatálisis , Dominio Catalítico , Cristalografía por Rayos X , Células Dendríticas/metabolismo , Células Dendríticas/virología , Nucleótidos de Desoxiadenina/metabolismo , Nucleótidos de Desoxicitosina/metabolismo , Nucleótidos de Desoxiguanina/metabolismo , Humanos , Hidrólisis , Modelos Biológicos , Modelos Moleculares , Proteínas de Unión al GTP Monoméricas/genética , Células Mieloides/virología , Nucleósido-Trifosfatasa/genética , Estructura Terciaria de Proteína , Transcripción Reversa , Proteína 1 que Contiene Dominios SAM y HD , Nucleótidos de Timina/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Replicación ViralRESUMEN
The SNF1 protein kinase complex plays an essential role in regulating gene expression in response to the level of extracellular glucose in budding yeast. SNF1 shares structural and functional similarities with mammalian AMP-activated protein kinase. Both kinases are activated by phosphorylation on a threonine residue within the activation loop segment of the catalytic subunit. Here we show that ADP is the long-sought metabolite that activates SNF1 in response to glucose limitation by protecting the enzyme against dephosphorylation by Glc7, its physiologically relevant protein phosphatase. We also show that the regulatory subunit of SNF1 has two ADP binding sites. The tighter site binds AMP, ADP, and ATP competitively with NADH, whereas the weaker site does not bind NADH, but is responsible for mediating the protective effect of ADP on dephosphorylation. Mutagenesis experiments suggest that the general mechanism by which ADP protects against dephosphorylation is strongly conserved between SNF1 and AMPK.
Asunto(s)
Adenosina Difosfato/metabolismo , Activación Enzimática/genética , Glucosa/metabolismo , Proteína Fosfatasa 1/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Transducción de Señal , Adenosina Difosfato/química , Adenilato Quinasa/genética , Adenilato Quinasa/metabolismo , Secuencia de Aminoácidos , Dominio Catalítico/genética , Secuencia Conservada , Regulación Fúngica de la Expresión Génica/fisiología , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Dominios y Motivos de Interacción de Proteínas , Proteína Fosfatasa 1/genética , Proteínas Serina-Treonina Quinasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Especificidad por Sustrato , Treonina/metabolismoRESUMEN
The isolation of broadly neutralizing antibodies against influenza A viruses has been a long-sought goal for therapeutic approaches and vaccine design. Using a single-cell culture method for screening large numbers of human plasma cells, we isolated a neutralizing monoclonal antibody that recognized the hemagglutinin (HA) glycoprotein of all 16 subtypes and neutralized both group 1 and group 2 influenza A viruses. Passive transfer of this antibody conferred protection to mice and ferrets. Complexes with HAs from the group 1 H1 and the group 2 H3 subtypes analyzed by x-ray crystallography showed that the antibody bound to a conserved epitope in the F subdomain. This antibody may be used for passive protection and to inform vaccine design because of its broad specificity and neutralization potency.
Asunto(s)
Anticuerpos Neutralizantes/inmunología , Anticuerpos Antivirales/inmunología , Antígenos Virales/inmunología , Glicoproteínas Hemaglutininas del Virus de la Influenza/inmunología , Virus de la Influenza A/inmunología , Animales , Anticuerpos Neutralizantes/aislamiento & purificación , Anticuerpos Antivirales/aislamiento & purificación , Especificidad de Anticuerpos , Células Cultivadas , Reacciones Cruzadas , Cristalografía por Rayos X , Epítopos/inmunología , Hurones , Glicosilación , Humanos , Interacciones Hidrofóbicas e Hidrofílicas , Inmunización Pasiva , Región Variable de Inmunoglobulina/inmunología , Subtipo H1N1 del Virus de la Influenza A/inmunología , Virus de la Influenza B/inmunología , Gripe Humana/inmunología , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Infecciones por Orthomyxoviridae/terapia , Células Plasmáticas/inmunología , Multimerización de Proteína , Estructura Secundaria de ProteínaRESUMEN
The heterotrimeric AMP-activated protein kinase (AMPK) has a key role in regulating cellular energy metabolism; in response to a fall in intracellular ATP levels it activates energy-producing pathways and inhibits energy-consuming processes. AMPK has been implicated in a number of diseases related to energy metabolism including type 2 diabetes, obesity and, most recently, cancer. AMPK is converted from an inactive form to a catalytically competent form by phosphorylation of the activation loop within the kinase domain: AMP binding to the γ-regulatory domain promotes phosphorylation by the upstream kinase, protects the enzyme against dephosphorylation, as well as causing allosteric activation. Here we show that ADP binding to just one of the two exchangeable AXP (AMP/ADP/ATP) binding sites on the regulatory domain protects the enzyme from dephosphorylation, although it does not lead to allosteric activation. Our studies show that active mammalian AMPK displays significantly tighter binding to ADP than to Mg-ATP, explaining how the enzyme is regulated under physiological conditions where the concentration of Mg-ATP is higher than that of ADP and much higher than that of AMP. We have determined the crystal structure of an active AMPK complex. The structure shows how the activation loop of the kinase domain is stabilized by the regulatory domain and how the kinase linker region interacts with the regulatory nucleotide-binding site that mediates protection against dephosphorylation. From our biochemical and structural data we develop a model for how the energy status of a cell regulates AMPK activity.
Asunto(s)
Proteínas Quinasas Activadas por AMP/química , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Difosfato/farmacología , Proteínas Quinasas Activadas por AMP/genética , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/farmacología , Regulación Alostérica/efectos de los fármacos , Regulación Alostérica/genética , Animales , Sitios de Unión , Cristalografía por Rayos X , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Cinética , Magnesio/metabolismo , Mamíferos , Modelos Moleculares , Fosforilación/efectos de los fármacos , Fosforilación/genética , Unión Proteica , Estructura Terciaria de Proteína/efectos de los fármacos , Estructura Terciaria de Proteína/genética , TermodinámicaRESUMEN
Lentiviruses are widespread in a variety of vertebrates, often associated with chronic disease states. However, until the recent discovery of the prehistoric endogenous lentiviruses in rabbits (RELIK) and lemurs (PSIV), it was thought that lentiviruses had no capacity for germline integration and were only spread horizontally in an exogenous fashion. The existence of RELIK and PSIV refuted these ideas, revealing lentiviruses to be present in a range of mammals, capable of germline integration, and far more ancient than previously thought. Using Gag sequences reconstructed from the remnants of these prehistoric lentiviruses, we have produced chimeric lentiviruses capable of infecting nondividing cells and determined structures of capsid domains from PSIV and RELIK. We show that the structures from these diverse viruses are highly similar, containing features found in modern-day lentiviruses, including a functional cyclophilin-binding loop. Together, these data provide evidence for an ancient capsid-cyclophilin interaction preserved throughout lentiviral evolution.
Asunto(s)
Proteínas de la Cápside/química , Ciclofilina A/metabolismo , Retrovirus Endógenos/química , Retrovirus Endógenos/genética , Evolución Molecular , Lentivirus/química , Lentivirus/genética , Animales , Secuencia de Bases , Cápside/metabolismo , Proteínas de la Cápside/genética , Proteínas de la Cápside/metabolismo , Cristalografía por Rayos X , Ciclofilina A/química , Metilación de ADN , Retrovirus Endógenos/fisiología , Productos del Gen gag/química , Productos del Gen gag/metabolismo , Genes Virales , Genes gag , Lemur/virología , Lentivirus/fisiología , Lentivirus de los Primates/química , Lentivirus de los Primates/genética , Lentivirus de los Primates/fisiología , Modelos Moleculares , Estructura Terciaria de Proteína , Conejos , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Virión/metabolismoAsunto(s)
Proteínas de Ciclo Celular/química , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/química , Secuencia de Aminoácidos , Sitios de Unión , Proteínas de Ciclo Celular/genética , Cristalografía por Rayos X , Modelos Moleculares , Datos de Secuencia Molecular , Pliegue de Proteína , Multimerización de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homología de Secuencia de Aminoácido , Electricidad Estática , Factores de Transcripción/genéticaRESUMEN
Clostridium difficile is a nosocomial bacterial pathogen causing antibiotic-associated diarrhea and fatal pseudomembranous colitis. Key virulence factors are toxin A and toxin B (TcdB), two highly related toxins that are members of the large clostridial toxin family. These large multifunctional proteins disrupt cell function using a glucosyltransferase domain that is translocated into the cytosol after vesicular internalization of intact holotoxin. Although substantial information about the biochemical mechanisms of intoxication exists, research has been hampered by limited structural information, particularly of intact holotoxin. Here, we used small-angle X-ray scattering (SAXS) methods to obtain an ab initio low-resolution structure of native TcdB, which demonstrated that this molecule is monomeric in solution and possesses a highly asymmetric shape with a maximum dimension of approximately 275 A. Combining this SAXS information with crystallographic or modeled structures of individual functional domains of TcdB reveals for the first time that the three-dimensional structure of TcdB is organized into four distinct structural domains. Structures of the N-terminal glucosyltransferase, the cysteine protease, and the C-terminal repeat region can be aligned within three domains of the SAXS envelope. A fourth domain, predicted to be involved in the translocation of the glucosyltransferase, appears as a large solvent-exposed protrusion. Knowledge of the shapes and relative orientations of toxin domains provides new insight into defining functional domain boundaries and provides a framework for understanding how potential intra-domain interactions enable conformational changes to propagate between domains to facilitate intoxication processes.
Asunto(s)
Proteínas Bacterianas/química , Toxinas Bacterianas/química , Clostridioides difficile/química , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/toxicidad , Toxinas Bacterianas/genética , Toxinas Bacterianas/toxicidad , Clostridioides difficile/genética , Clostridioides difficile/patogenicidad , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Secuencias Repetitivas de Aminoácido , Dispersión del Ángulo Pequeño , Homología de Secuencia de Aminoácido , Homología Estructural de Proteína , Difracción de Rayos XRESUMEN
The Mre11/Rad50/Nbs1 protein complex plays central enzymatic and signaling roles in the DNA-damage response. Nuclease (Mre11) and scaffolding (Rad50) components of MRN have been extensively characterized, but the molecular basis of Nbs1 function has remained elusive. Here, we present a 2.3A crystal structure of the N-terminal region of fission yeast Nbs1, revealing an unusual but conserved architecture in which the FHA- and BRCT-repeat domains structurally coalesce. We demonstrate that diphosphorylated pSer-Asp-pThr-Asp motifs, recently identified as multicopy docking sites within Mdc1, are evolutionarily conserved Nbs1 binding targets. Furthermore, we show that similar phosphomotifs within Ctp1, the fission yeast ortholog of human CtIP, promote interactions with the Nbs1 FHA domain that are necessary for Ctp1-dependent resistance to DNA damage. Finally, we establish that human Nbs1 interactions with Mdc1 occur through both its FHA- and BRCT-repeat domains, suggesting how their structural and functional interdependence underpins Nbs1 adaptor functions in the DNA-damage response.
Asunto(s)
Proteínas de Ciclo Celular/química , Proteínas Cromosómicas no Histona/química , Reparación del ADN , Proteínas Nucleares/química , Proteínas de Schizosaccharomyces pombe/química , Schizosaccharomyces/química , Secuencia de Aminoácidos , Cristalografía por Rayos X , Daño del ADN , Proteínas de Unión al ADN/metabolismo , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Fosforilación , Estructura Terciaria de Proteína , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Alineación de SecuenciaRESUMEN
The viruses that caused the three influenza pandemics of the twentieth century in 1918, 1957, and 1968 had distinct hemagglutinin receptor binding glycoproteins that had evolved the capacity to recognize human cell receptors. We have determined the structure of the H2 hemagglutinin from the second pandemic, the "Asian Influenza" of 1957. We compare it with the 1918 "Spanish Influenza" hemagglutinin, H1, and the 1968 "Hong Kong Influenza" hemagglutinin, H3, and show that despite its close overall structural similarity to H1, and its more distant relationship to H3, the H2 receptor binding site is closely related to that of H3 hemagglutinin. By analyzing hemagglutinins of potential H2 avian precursors of the pandemic virus, we show that the human receptor can be bound by avian hemagglutinins that lack the human-specific mutations of H2 and H3 pandemic viruses, Gln-226Leu, and Gly-228Ser. We show how Gln-226 in the avian H2 receptor binding site, together with Asn-186, form hydrogen bond networks through bound water molecules to mediate binding to human receptor. We show that the human receptor adopts a very similar conformation in both human and avian hemagglutinin-receptor complexes. We also show that Leu-226 in the receptor binding site of human virus hemagglutinins creates a hydrophobic environment near the Sia-1-Gal-2 glycosidic linkage that favors binding of the human receptor and is unfavorable for avian receptor binding. We consider the significance for the development of pandemics, of the existence of avian viruses that can bind to both avian and human receptors.